In a basic example of hybridization, nucleic acid probes or primers are designed to bind, or “hybridize,” with a target nucleic acid, for example RNA, in a sample. One type of hybridization application, in situ hybridization (ISH), includes hybridization to a target in a specimen wherein the specimen may be in vivo, in situ, or for example, fixed or adhered to a glass slide (i.e., in vitro). Probes may then be used, for example, to detect genetic abnormalities in a target sequence, providing valuable information about, e.g., prenatal disorders, cancer, and other genetic or infectious diseases.
The efficiency and accuracy of nucleic acid hybridization assays mostly depend on at least one of three major factors: a) denaturation (i.e., separation of, e.g., two nucleic acid strands) conditions, b) renaturation (i.e., re-annealing of, e.g., two nucleic acid strands) conditions, and c) post-hybridization washing conditions.
In order for the probes or primers to bind to the target nucleic acid in the sample, complementary strands of nucleic acid must be separated (i.e., denatured). Although RNA is a single-stranded molecule and, therefore, should not require a strand-separation step in order to bind to the primer/probe, RNA molecules can pair with single-stranded DNA molecules or with other RNA molecules. These associations are stabilized by hydrogen bonding between bases on opposite strands when bases are paired in a particular way (A+T/U or G+C) and by hydrophobic bonding among the stacked bases. In addition, complementary base-pairing and other types of hydrogen bonds can occur between nucleotides in the same RNA molecule, causing parts of the RNA to fold and pair with itself in a double helical configuration. Thus, some RNA hybridization applications may optionally include a denaturation step.
Traditional hybridization experiments, such as ISH assays, use high temperatures (e.g., 95° C. to 100° C.) and/or formamide-containing solutions to denature doubled stranded nucleic acid. Formamide disrupts base pairing by displacing loosely and uniformly bound hydrate molecules and by causing “formamidation” of the Watson-Crick binding sites. Thus, formamide has a destabilizing effect on double stranded nucleic acids and analogs. However, formamide is a toxic, hazardous material, subject to strict regulations for use and waste. In addition, the use of high formamide concentrations appears to cause morphological destruction of cellular, nuclear, and/or chromosomal structure. Heat can also be destructive to the sample because the phosphodiester bonds of the nucleic acids may be broken at high temperatures, leading to a collection of broken single stranded nucleic acids. In addition, heat can lead to complications when small volumes are used, since evaporation of aqueous buffers is difficult to control.
Once any double-stranded nucleic acids have been separated, a “renaturation” or “reannealing” step allows the primers or probes to bind to the target nucleic acid in the sample. This step is also sometimes referred to as the “hybridization” step. The re-annealing step is by far the most time-consuming aspect of traditional hybridization applications. See FIGS. 1 and 2 (presenting examples of traditional hybridization times for DNA templates). In addition, the presence of formamide in a hybridization buffer can significantly prolong the renaturation time, as compared to aqueous denaturation solutions without formamide.
After the probe has annealed to the target nucleic acid in the sample, any unbound and mis-paired probe is removed by a series of post-hybridization washes. The specificity of the interaction between the probe and the target is largely determined by stringency of these post-hybridization washes. Duplexes containing highly complementary sequences are more resistant to high-stringency conditions than duplexes with low complementary. Thus, increased stringency conditions can be used to remove non-specific bonds between the probe and the target nucleic acids. Four variables are typically adjusted to influence the stringency of the post-hybridization washes: (1) temperature (as temperature increases, non-perfect matches between the probe and the target sequence will denature, i.e., separate, before more perfectly matched sequences); (2) salt conditions (as salt concentration decreases, non-perfect matches between the probe and the target sequence will denature, i.e., separate, before more perfectly matched sequences); (3) formamide concentration (as the amount of formamide increases, non-perfect matches between the probe and the target sequence will denature, i.e., separate, before more perfectly matched sequences); and (4) time (as the wash time increases, non-perfect matches between the probe and the target sequence will denature, i.e., separate, before more perfectly matched sequences).
The present invention provides several potential advantages over prior art methods and compositions for RNA hybridization applications. These advantages include faster hybridization times, lower hybridization temperatures, and less toxic hybridization solvents.